Monocytes, macrophages, and microglia are mononuclear phagocytes important in innate immunity, but also key reservoirs of HIV in the central nervous system. These reservoirs represent a challenge to HIV-1 eradication since they remain producing virus in tissue despite the presence of antiretroviral therapy. Cystatin B expression is positively correlated with HIV replication and decreased levels STAT-1 phosphorylation (STAT-1PY) in monocyte-derived macrophages (MDM). However, the players and the mechanism by which this occurs are unknown. This work is expected to elucidate the mechanism by which cystatin B contributes to HIV-1 replication by regulation of STAT-1PY in macrophage reservoirs. The objective of in this particular application is to define the relationships and signaling pathways between cystatin B, STAT-1PY, interferon (IFN), and HIV persistence in MDM. To attain the objective of this proposal, we will test hypothesis that cystatin B promotes HIV persistence by interacting with the STAT-1PY and additional proteins, including those related to IFN signaling pathway. This hypothesis has been formulated on the basis of preliminary data produced in the applicants' laboratories. The rationale of the proposed research is that understanding the role of cystatin B in HIV replication will permit the modulation of this protein or its interacting proteins and decrease HIV within macrophage reservoirs. Guided by strong preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) Define the cystatin B/STAT-1 signaling pathways and proteins associated to STAT-1 phosphorylation and HIV replication in macrophages; and 2) Identify the role of Cystatin B in the JAK/STAT-1 pathway during HIV infection and IFN-2 activation. Under the first aim we will use In situ Proximity Ligation Assay (PLA) (Olink Biosciences) to confirm, localize and quantify this protein interaction in uninfected and HIV infected MDM and determine if cystatin B interacts directly or indirectly with STAT-1. We will also identify the specific protein pathways associated to cystatin that are subject to modification by phosphorylation using spectral counting based quantification. Under the second aim, we will determine the effect of cystatin B in LTR mediated HIV replication and IFN induced antiviral response using the luciferase assay and correlate our findings with what happens in the MDM. This approach is innovative because, the role of cystatin B as a regulatory protein for in HIV replication has not been demonstrated using proteomics approaches and the interacting partners have not been found. The proposed research is significant because it will reveal novel mechanisms of HIV persistence that could be targeted for new therapeutic approaches directed to eliminate HIV in macrophage reservoirs.